白藜芦醇调控Wnt信号通路增强替莫唑胺抗脑胶质瘤作用的体内研究

In vivo study of resveratrol regulating Wnt signaling pathway to enhance temozolomide against gliomas

目的探讨白藜芦醇于脑胶质瘤模型体内增强替莫唑胺抗脑胶质瘤作用的分子机制。方法将人源性恶性胶质瘤细胞系T98G移植入BALB/C-nu雌性裸鼠中构建人脑胶质瘤细胞裸鼠原位移植模型。造模5 d后将造模成功的48只裸鼠按随机数字表法分为溶剂对照组、白藜芦醇组、替莫唑胺组、联合用药组、Wnt信号通路激动剂组、Wnt信号通路抑制剂组,每组8只,并分别予二甲基亚砜10 mg/kg、白藜芦醇10 mg/kg、替莫唑胺25 mg/kg、白藜芦醇10 mg/kg+替莫唑胺25 mg/kg、白藜芦醇10 mg/kg+替莫唑胺25 mg/kg+氯化锂2 mg/kg、白藜芦醇10 mg/kg+替莫唑胺25 mg/kg+IWR-15 mg/kg腹腔注射,1次/d,持续30 d。给药期间持续观察各组裸鼠的生存状况,每隔5天采用MRI检测肿瘤体积。给药30 d后,采用TUNEL染色检测肿瘤细胞的凋亡情况,采用免疫荧光染色检测肿瘤组织中O6-甲基鸟嘌呤-DNA甲基转移酶(MGMT)、β-连环蛋白的免疫荧光强度,采用Western blotting检测Wnt信号通路相关蛋白(Wnt2、β-连环蛋白)、MGMT、糖原合成激酶3β(GSK3β)蛋白的表达水平。结果肿瘤体积检测显示:与替莫唑胺组相比,联合用药组、Wnt信号通路抑制剂组造模后第20、25、30、35天的肿瘤体积均明显减小,差异均有统计学意义(P<0.05)。与联合用药组相比,Wnt信号通路抑制剂组造模后第20、25、30、35天的肿瘤体积均明显减小,Wnt信号通路激动剂组造模后第20、25、30、35天的肿瘤体积均明显升高,差异均有统计学意义(P<0.05)。TUNEL染色显示:与替莫唑胺组相比,联合用药组、Wnt信号通路抑制剂组的肿瘤细胞凋亡率均明显升高,差异均有统计学意义(P<0.05)。与联合用药组相比,Wnt信号通路抑制剂组的肿瘤细胞凋亡率明显升高,Wnt信号通路激动剂组的肿瘤细胞凋亡率明显降低,差异均有统计学意义(P<0.05)。免疫荧光染色显示:与联合用药组相比,Wnt信号通路抑制剂组中MGMT、β-连环蛋白的免疫荧光强度明显降低,Wnt信号通路激动剂组中MGMT、β-连环蛋白的免疫荧光强度明显增强。Western blotting检测显示:与联合用药组相比,Wnt信号通路抑制剂组中Wnt2、β-连环蛋白和MGMT的蛋白表达均明显降低,GSK-3β的蛋白表达明显升高;Wnt信号通路激动剂组中Wnt2、β-连环蛋白和MGMT的蛋白表达均明显升高,GSK-3β的蛋白表达明显降低,差异均有统计学意义(P<0.05)。结论白藜芦醇通过降低Wnt2、β-连环蛋白的表达抑制Wnt信号通路,导致MGMT表达下降,从而增强替莫唑胺抗脑胶质瘤作用。

Objective To investigate the effect of resveratrol(Res)on temozolomide(TEM)against gliomas in vivo.Methods Human glioma cell line T98G was transplanted into BALB/C-nu female nude mice to establish orthotopic human glioma cell transplanted models.Five d after modeling,the 48 successfully modeled nude mice were randomly divided into solvent control group,Res group,TEM group,combination drug group,Wnt signaling pathway agonist group,and Wnt signaling pathway inhibitor group(n=8);and dimethyl sulfoxide(10 mg/kg),Res(10 mg/kg),TEM(25 mg/kg),Res(10 mg/kg+TEM(25 mg/kg),Res(10 mg/kg)+TEM(25 mg/kg)+lithium chloride(2 mg/kg),and Res(10 mg/kg)+TEM(25 mg/kg)+IWR-1(5 mg/kg)were given,respectively,once/d for 30 d.During the administration,the survival status of nude mice in each group was continuously observed,tumor volume was measured by MR imaging every 5 d.Thirty d after administration,TUNEL was used to detect the apoptosis of tumor cells,and immunofluorescence was used to detect the immunofluorescent intensity of O6-methylguanine-DNA methyltransferase(MGMT)andβ-catenin in the tumor tissues.Western blotting was used to detect the protein expression levels of Wnt signaling pathway-related proteins(Wnt2,andβ-catenin),MGMT,and glycogen synthase kinase 3β(GSK3β).Results As compared with the TEM group,the combination drug group and Wnt signaling pathway inhibitor group had significantly decreased tumor volumes 20,25,30,and 35 d after modeling(P<0.05);as compared with the combination drug group,the Wnt signaling pathway inhibitor group had significantly decreased tumor volumes while Wnt signaling pathway agonist group had significantly increased tumor volumes 20,25,30,and 35 d after modeling(P<0.05).TUNEL showed that the apoptosis rate of tumor cells in the combination drug group and Wnt signaling pathway inhibitor group was significantly increased as compared with that in the temozolomide group(P<0.05);as compared with that in the TEM group,the apoptosis rate of tumor cells in the Wnt signaling pathway inhibitor group was significantly increased while that in the Wnt signaling pathway agonist group was statistically decreased(P<0.05).Western blotting results showed that as compared with those in the combination drug group,the protein expression levels of Wnt2,β-catenin,and MGMT in the Wnt signaling pathway inhibitor group were significantly reduced,and GSK-3βprotein expression level was significantly increased;while the protein expression levels of Wnt2,β-catenin,and MGMT in the Wnt signaling pathway agonist group were significantly increased,and GSK-3βprotein expression level was significantly decreased(P<0.05).Conclusion Res inhibits Wnt signaling pathway by reducing expressions of Wnt2 andβ-catenin,leading to decrease in MGMT expression,thereby enhancing the anti-glioma effect of TEM.